Due to their unique properties, nano-sized carbon materials are predicted to have numerous applications in industry, but significant evidence exists to suggest their potential to cause toxicity. To determine if pre-exposure to side-stream cigarette smoke (SSCS) influences their toxicity, we examined the pulmonary response of smoke-exposed mice to multi-walled carbon nanotubes (MWCNT). Female A/J mice were exposed to SSCS in a whole body exposure chamber at approximately 40 mg/m(3) for 4 weeks (6 h/d, 5 d/wk) and challenged with a single dose of MWCNT (40 µg) by the pharyngeal aspiration technique. A total of four groups were compared: air/phosphate buffered saline (PBS)-control, SSCS/PBS, air/MWCNT, and SSCS/MWCNT. At days 1 and 3 post-MWCNT treatment, lung tissues and bronchoalveolar lavage fluid (BALF) were collected and analyzed. In comparison with controls, significantly higher levels of total BAL cells were obtained from mice exposed to SSCS and MWCNT alone or combination. Influx of polymorphonuclear leukocytes (PMN) into BALF greatly increased in MWCNT alone and SSCS/MWCNT groups at both days 1 and 3 compared with controls. However, pre-exposure to SSCS significantly suppressed PMN response to MWCNT on day 1 but not day 3. Total BALF protein, lactate dehydrogenase, and mucin were significantly elevated in MWCNT and SSCS/MWCNT groups but not in the SSCS group, except mucin at day 3, when compared with controls. These results demonstrate that MWCNT markedly increases pulmonary toxicity in mice and SSCS pre-exposure plays a minor role in modulating MWCNT-induced lung toxicity at the concentrations and time points selected in the present study.